PSCs are defined as cell entities which have both the capacity to differentiate toward multiple cell lineages non-identical to themselves (pluripotency) and a capability of producing a cell having an identical capacity to the originated cell as a daughter cell when they divide (self-renewal), which is considered to include embryonic stem (ES) cell and induced pluripotent stem (iPS) cell. And because of these characteristics, ES cell and iPS cell are strongly anticipated to play important roles in regenerative medicine and/or drug screening.
However, it has been recently reported that mouse PSCs and human PSCs do not share some of their fundamental properties and moreover, human PSCs are generally considered to have lower differentiation capabilities when compared to mouse PSCs.
Mouse PSCs can be challenged experimentally for their pluripotency with some accuracy. For example, mouse ES cells can be injected into a cavity of blastocyst where the injected cells intermingle with the host inner cell mass (ICM), followed by a normal development of ES cell-derived cells in an embryo, which is called as chimera formation. The fact that mouse ES cells can synchronize their development with a timing-matched ICM is a good verdict for pluripotency. In this case, as development further proceeds, it is known that part of the injected ES cells might also contribute to the germline. This germline transmission means that these cells can contribute to the next generation and further corroborates the compatibility as normal cells in a more generic term.
There also exists an “ultimate” way to show pluripotency. When we inhibit the first cell cleavage of a mouse fertilized egg transiently and culture it in a culture dish, we can obtain a tetraploid blastocyst. The tetraploid blastocyst looks almost normal but will not continue development into embryo further. Therefore, just by putting tetraploid embryos back to a uterus of pseudo-pregnant female mouse would not produce a live pup. But if we inject stem cells with differentiation capabilities into these tetraploid blastocysts and let them contribute to the chimera formation as mentioned above, the PSCs now would be fully responsible to contribute to development as they are the sole cells with normal karyotype. In this way, we can obtain a completely PSC-derived body in a single generation. This methodology is called the tetraploid-complementation assay and the obtained pups are called as all-iPS mice, according to the PSCs injected. This multiple tests enable strict assessment of pluripotencies in mouse and mouse iPS cells have been shown to exhibit strict pluripotencies using the battery of such multiple tests.
In sharp contrast, human pluripotencies cannot be scrutinized in these fashions because of ethical issues. An alternative way to show pluripotency in human contexts is teratoma formation. When stem cells are transplanted into nude mice subcutaneously, these cells when supported by the host blood supply, do differentiate at random to form an organ. The formed stem cell-derived tumor mass will be pathologically examined to find ectoderm, mesoderm and endoderm, and thereby, the originated stem cells are qualified as pluripotent. However, this methodology whereby we assess pluripotency by teratoma formation is inherently problematic. That is, even if the original stem cells have insufficient efficacy of differentiation, any differentiation efficacy above zero could be judged as “pluripotency” if three germ layers are observed. Teratoma assay would judge the pluripotency without considering the existence of residual undifferentiated cells within the teratoma. With the current paradigm of teratoma assay, the mere presence of a limited number of bona fide pluripotent cells within the cell population would be sufficient and the current teratoma assay has no implication about the quantitative trait of pluripotency. For example, mouse iPS cells derived using c-Myc, albeit showing three-germ-layer differentiation in teratoma assays, were prone to give iPS cell-derived tumors upon chimera formation. An alternative to teratoma assay would be to independently direct cell differentiation toward the three germ layers. However, there is no “golden standard” in any cell differentiation protocol making this strategy inapplicable for quantitative analysis.
There is also a more profound and fundamental problem with human PSCs. This is about the possible absence of an ideal PSC for human which bears the capacity to be amenable to equally robust differentiation toward all the cell lineages. Osafune et al. have reported that no two human ES cell lines showed quasi-identical gene expression profile, and thus each ES cell line has own propensity to differentiate into a specific germ layer, and further, suggested that human ES cells do not have sufficient pluripotency in terms of quantitative evaluation (non-patent literature 1).
It took 17 years after the discovery of mouse ES cell to establish human ES cell. In retrospect, the major reason for this delay was that human ES cell could not be established in the same way as in the mouse ES cell. Although mouse ES cells are dependent to leukemia inhibitory factor (LIF) for their self renewal, human ES cell cannot show self renewal ability in a medium added with LIF. Thomson et al. found that human ES cell requires fibroblast growth factor (FGF) and Activin/Nodal for its maintenance (non-patent literature 2). These differential requirements for mouse and human ES cell self renewal diversify into different intracellular signaling pathways: JAK/STAT signaling and SMAD2/3 signaling for mouse and human ES cell, respectively. The currently major view is that SMAD2/3 signaling in human PSCs activates the expression of NANOG. In parallel, SMAD2/3 signaling is a critical factor for the mesendoderm development in mammals and therefore a high concentration of Activin is required for proper mesendoderm differentiation of PSCs. It is therefore interesting to note that the current human PSC self renewal signaling crossovers with the signal which would induce its mesendodermal differentiation.
Mouse and human ES cells both originate from blastocysts. However, most probably owing to their difference in the culture conditions, their characteristics significantly differ. A major contrast is albeit mouse ES cells retain characteristics of their originated blastocyst inner cells, human ES cells are more akin to the primitive ectoderm of the epiblast, which appears after some development of the blastocyst. Then, the epiblast stem cell (EpiSC) was established directly from mouse epiblasts using culture condition equivalent to human ES cells (non-patent literature 3 and 4). This had led the stem cell biologists to discriminate two stages of pluripotency. One is the blastocyst-type pluripotency, represented by mouse ES as well as iPS cells and the other is the epiblast-type pluripotency, which includes human ES, human iPS cells and mouse EpiSCs. The former pluripotency is now called “naive” and the latter “primed”, originally coined by Smith et al. (non-patent literature 5).
Naive and primed pluripotencies share the three germ layer differentiation capabilities and teratoma assay-compatibilities upon transplantation into nude mouse. However, naive PSCs are more similar to the earlier stage (blastocyst) of development closer to fertilized ovum, are easier to be established in culture and possess fuller pluripotency in that an individual body completely derived from these cells can be generated. In contrast, primed PSCs are more akin to the epiblast, an embryonic stage following the blastocyst, and are empirically tougher to establish. Additionally, as described above, primed PSCs have clear differentiation propensities (non-patent literature 1). Finally, it has been recognized that primed PSCs poorly synchronize their development with early embryo and therefore do not produce chimera, and thus it is known that primed human PSCs are inferior to mouse naive PSCs in differentiation efficacy including pluripotency.
In practicing regenerative medicine and drug development, human PSCs are being anticipated to exert their high differentiation capabilities in order to supply various cell types, tissues and organogenesis. In this line, it seems desirable to enhance differentiation efficacy of primed human PSCs. However, this kind of innovation remains to be developed.